This application requests continued support to further define the role of SP-A in pulmonary biology and surfactant homeostasis. The specific aims are designed to test two major hypotheses. The first aim will test whether the temporal-spatial expression of SP-A is mediated by cis-active elements that interact with cell selective nuclear transcriptional proteins that determine SP-A gene expression in distinct subsets of respiratory epithelial cells. Transcriptional and post-transcriptional events modulating SP-A gene expression will be discerned utilizing in vitro assays to identify protein-DNA or protein-RNA interactions. To confirm that identified sequences serve as transcriptional and post- transcriptional regulators of SP-A synthesis, cis-active regions will be tested in cell transfection studies in vitro and in transgenic mice in vivo. Regions of the SP-A gene that determine lung cell specificity will be identified, modified by site-specific mutagenesis and further tested for their interactions with nuclear protein by gel retardation, DNase footprinting and by binding to nuclear proteins (including members of the AP1 HNF-3, TTF-1 and novel transcriptional proteins) that may determine the temporal-spatial distribution of SP-A expression in the lung. A second major aim will test the hypothesis that SP-A plays a critical role in lung function, including surfactant recycling, secretion, surface activity of phospholipids, and host defense. This latter aim will be dependent upon the generation of transgenic mice with null mutation in the SP-A gene locus. Heterozygote and homozygote mice are being produced by gene ablation. These mice will be used for physiologic and biochemical studies, to further define the role of SP-A in lung function in health and disease, and to discern the primary genetic mechanisms that control SP-A gene expression in a lung-epithelial selective manner. Elucidation of the role of SP-A and the genetic elements controlling SP-A expression will be critical for development of improved therapies for respiratory distress syndrome in premature infants, and for surfactant replacement therapy in acute lung injuries associated with alveolar collapse or infection.